Roles of lipid raft in the regulation of sperm motility in the ascidian Ciona intestinalis

Thesis (Ph. D. in Science)--University of Tsukuba, (A), no. 5938, 2011.11.30Includes bibliographical references (p. 60-75

The Roles of Primary Cilia in Cardiovascular Diseases and Cancer

Primary cilia are sensory organelles present in most mammalian cell types and regulate cell cycle and signaling pathways. Biochemical and molecular dysfunctions of primary cilia are associated with a wide range of diseases, including cancer, ciliopathies polycystic kidney disease (PKD, liver disorders, mental retardation, and obesity to cardiovascular diseases. Dysfunction in endothelial cilia contributes to aberrant fluid-sensing and results in vascular disorders, such as hypertension, aneurysm, and atherosclerosis. In this dissertation, the most recent outcomes on the roles of endothelial primary cilia within vascular biology have been summarized. Moreover, we evaluate the correlation between cilia formation or length and cell cycle or division using PKD and cancer epithelia. The results show that these cells were associated with abnormal ploidy and were highly proliferative compared with normal kidney epithelia (NK). Importantly, the cancer epithelial cells show a reduction in the presence and/or length of primary cilia. Restoration of the expression and length of primary cilia in these cells using rapamycin were inversely correlated with cell proliferation. Our data suggest that primary cilia may serve as a novel target in cardiovascular disorder and cancer

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Studies of human genetic diseases and developmental processes with the frog Xenopus laevis

Next generation sequencing is a driving force behind the identification of genes and alleles that are suspected to cause human genetic diseases. In silico tools are routinely used in the clinical everyday life to characterize unknown genotypes. However, these tools have a limited predictive accuracy and can only provide a first-line assessment. Especially un- or less studied genes require in every case predictive in vivo model systems that allow conclusions about disease associations. Classically, mice and zebrafish are utilized for such research, which concomitantly deepens the understanding of the involved developmental processes. In this collection of studies, the African clawed frog Xenopus laevis was used to explore and promote its suitability for the analysis of potential human disease genes, variants and their associated developmental processes. The first chapters covers potential candidate genes for primary ciliary dyskinesia (PCD). The second chapter addresses if an actin based motor protein and a novel metzincin peptidase, encoded by myosin ID (MYO1D) and leishmanolysin like peptidase (LMLN2)/tout-de-travers (TDT), respectively, are potentially causative for PCD independent laterality defects. The third chapter deals with two candidates for neurodevelopmental disorders, namely hyaluronan mediated motility receptor (HMMR) and progesterone immunomodulatory binding factor 1 (PIBF1).Next generation sequencing ist eine treibende Kraft hinter der Identifizierung von Genen und Allelen, die mutmaßlich humane Erbkrankheiten verursachen. Im klinischen Alltag werden routinemäßig in silico Methoden eingesetzt, um unbekannte Genotypen zu charakterisieren. Diese Methoden haben jedoch eine limitierte Vorhersagegenauigkeit und ermöglichen nur eine erste Beurteilung. Besonders spärlich oder nicht analysierte Gene erfordern in jedem Fall aussagekräftige in vivo Modellsysteme, die Schlüsse über etwaige Krankheitsassoziationen zulassen. Klassischer Weise werden für solche Forschungsbemühungen, die beiläufig auch das Verständnis der daran beteiligten Entwicklungsprozesse vertieft, Mäuse oder Zebrabärblinge verwendet. In dieser hier vorliegenden Studienkollektion wurde der Afrikanische Krallenfrosch Xenopus laevis verwendet, um dessen Eignung für die Analyse potentieller humaner Krankheitsgene, -genvarianten und deren assoziierten Entwicklungsprozesse zu ergründen und dies herauszustellen. Im ersten Kapitel werden Kandidatengene der primären ciliären Dyskinesie (PCD) besprochen. Das zweite Kapitel behandelt die Frage ob ein Aktin basierendes Motorprotein und eine neue Metzinkin Peptidase, jeweils von myosin ID (MYO1D) und leishmanolysin like peptidase 2 (LMLN2)/tout-de-travers (TDT) codiert, potentiell für PCD unabhängige Lateralitätsdefekte verantwortlich sein könnten. Das dritte Kapitel befasst sich mit zwei Kandidaten für neurologische Entwicklungsstörungen, namentlich hyaluronan mediated motility receptor (HMMR) und progesterone immunomodulatory binding factor 1 (PIBF1)

PKD2-dependent sensing mechanism in Kupffer’s vesicle: how it affects the left-right axis establishment in zebrafish

RESUMO: Estabelecimento do eixo Esquerda-Direita (ED) é um processo complexo que ocorre cedo durante o desenvolvimento. Exige a integração de várias vias de sinalização, tais como TGF- β, Notch, Wnt e Cálcio. Também envolve a coordenação de dinâmica de fluídos, difusão de morfogénios e movimento ciliar. Tudo junto e coordenado no tempo leva à correta localização dos órgãos internos. Problemas no estabelecimento deste eixo estão normalmente associados a doenças crónicas. As primeiras decisões assimétricas acontecem numa pequena e transiente estrutura chamada o Organizador Esquerda Direita, uma estrutura ciliar que existe em vários vertebrados. Cílios móveis geram um fluxo de fluido assimétrico que se traduz numa expressão génica assimétrica entre o lado esquerdo e o lado direito. Estes sinais são depois transferidos à Mesoderme Lateral, o tecido que mais tarde dá origem ao coração e influencia a endoderme que dá origem a órgãos como o fígado e o pâncreas. No Capítulo 2, focámo-nos em tentar compreender como é que as vias de sinalização se coordenavam para decidir entre um cílio móvel e imóvel no Organizador. Embora a expressão de Foxj1a em todas as células do Organizador produza cílios com ultra-estrutura compatível com motilidade, a decisão entre mover ou não é exclusiva da via de sinalização Notch. A seguir, focámo-nos em caracterizar Pkd2, um canal de cálcio importante no estabelecimento do eixo ED. Este canal está associado ao Pkd1l1, uma molécula com domínios capazes de sentir fluido e responder com entrada de cálcio na célula. No Capítulo 3, questionámos se não ter fluxo no Organizador tinha o mesmo impacto que não ter um mecanismo para sentir esse fluxo através da ausência do Pkd2. A única manipulação que não afetou a arquitetura do Organizador foi remover o Pkd2 apenas nas células do Organizador, o que não é muito eficiente e tem impacto na velocidade do fluxo dentro do Organizador. Ainda assim, todas as manipulações efetuadas deram o mesmo fenótipo: randomização da posição dos órgãos. No Capítulo 4, fizemos um estudo de transcritómica usando apenas as células do Organizador em embriões normais ou injetados com morpholino contra o Pkd2. Embora não tenhamos encontrado genes com expressão assimétrica à volta do Organizador, encontramos quatro novos genes que influenciam o estabelecimento do eixo: cacybp, frzb, pvalb6 e ncl1. No Capítulo 5, focámo-nos na ncl1, um gene nunca antes associado ao estabelecimento do eixo e que atua como antagonista da via de sinalização TGF-β ao influenciar a secreção de Lefty e influenciando a padronização da mesoderme-endoderme. Nós descobrimos que este gene impactua no estabelecimento do eixo, provavelmente ao influenciar a secreção de algum elemento da via de sinalização TGF-β. No Capítulo 6, focámo-nos em estabelecer o peixezebra como um bom modelo para estudar toxicidade no rim em resposta a fármacos. Em suma, os resultados apresentados nesta tese providenciam novas pistas para o estabelecimento do eixo ED, desde o movimento do cílio a novos genes a jusante do Pkd2 e do cálcio. Também reforçámos a ideia de que o peixe-zebra pode ser um bom modelo para estudar doenças humanas.ABSTRACT: Left-Right (LR) axis establishment is a complex process that happens early in development. It requires the interplay of several genetic pathways like TGF-β, Notch, Wnt and Calcium signalling. It also involves the integration of fluid dynamics, morphogen diffusion and cilium biosynthesis to correctly position the internal organs in their final destinations. Problems in LR axis establishment are often associated with chronic diseases. The first asymmetric decision commonly happens in a small transient structure, the Left-Right Organizer (LRO), a ciliated structure present in many vertebrates. Motile cilia generate an asymmetric fluid flow that is perceived differently between the left and the right side, which generates a calcium response and asymmetric gene expression. These signals are then transferred to the Lateral Plate Mesoderm, the tissue that will later give rise to the heart and influence the endoderm derived organs such as the liver and pancreas. In Chapter 2, we focused in understand the pathways behind deciding between being a motile vs immotile cilium in the LRO. Although all cilia are made motile in terms of ultrastructure due to Foxj1a expression, the decision to move or not is dependent on Notch signalling alone. Then, we focused on further characterization of an important calcium channel, Pkd2, in the LR. This channel is thought to partner with Pkd1l1 and sense flow, an important feature in LR. In Chapter 3, we asked if having no flow had the same impact as having no Pkd2-mediated sensing. The only manipulation that did not affect the LRO architecture was to target Pkd2 on the LRO cells only, which still left visible Pkd2 protein and a slower flow. Still, all our manipulations had the same phenotype: randomization of organ situs. In Chapter 4, we performed a LRO-specific microarray between WT and pkd2 morphant embryos in order to find other asymmetric genes present in the LRO. Although we did not find any gene expressed asymmetrically between left and right side, we indeed find four new genes with minor roles in LR: cacybp, frzb, pvalb6 and ncl1. In Chapter 5, we further focused in manipulating ncl1, a new gene in LR that is known to act as a TGF-β antagonist by facilitating Lefty secretion and impacting on mesendoderm patterning. Indeed, we found that it has an impact on LR, probably by influencing the secretion of some TGF- β signalling player. In Chapter 6, we set to establish zebrafish as a good model to study kidney toxicity when metabolizing drugs. Together, the results presented in this thesis provide new clues for LR axis establishment, from cilia motility to new downstream genes of Pkd2 and calcium. It also highlights the zebrafish as a good model to study human disease

Axes determination in the frog Xenopus laevis : the function of the goosecoid, myo1d and dmrt2

During early embryogenesis, pattern formation processes along the head-trunk (anteroposterior, AP), belly-back (dorsoventral, DV) and left-right (LR) body axis generate the fundamental body plan of the bilateria. The formation of the LR axis is exceptional because externally our body is bilateral symmetric whereas most inner organs are shaped and positioned asymmetrically. The three body axes are basically specified during gastrulation and neurulation by a set of developmental control genes. The aim of this work was to analyze the function of the highly conserved genes, goosecoid (gsc), myosin1d (myo1d) und dmrt2 during body axis determination in Xenopus. The first chapter of this work describes the activity of the homeobox transcription factor Goosecoid during AP- and DV-axis formation. Gsc acts as an autoregulatory transcriptional repressor and importantly is expressed in the Spemann Organizer (SO) of all vertebrate embryos. The SO represents the main dorsal signaling center for primary axis induction, regulates embryonic patterning and cell movements. It is further required for AP i.e. head and trunk development. Transferring of SO or gsc misexpression to ventral half of embryos resultes in secondary axis formation i.e. siamnese twins. However, SO function of Gsc was enigmatic, as gsc mutants showed no defects on early developmental processes what challenged Gsc function in the SO. In this chapter, gsc was characterized by conducting gain of function experiments in the embryonic midline of Xenopus embryos. Gsc was able to repress planar cell polarity (PCP) in a cell- and non-cell autonomous fashion leading to neural tube closure defects. In the early gastrulae, Gsc separates the head from the trunk mesoderm by repressing the mesodermal t-box gene transcription factor T (Tbxt). This inhibition allows the migration of the head mesodermal cells whereas the trunk notochord elongates by mediolateral intercalation. Gsc activity on PCP signaling seems to be specific for vertebrates only and correlates with the presence of two novel domains. The determination of the LR body axis is discussed in the second chapter of this work. At the so called left-right organizer (LRO) a cilia-mediated leftward-fluid flow initiates the symmetry breaking event in neurulae embryos. Lateral sensory cells (sLRO) of the LRO perceive flow on the left side and translate it into the left asymmetric induction of the highly conserved Nodal cascade. If and how the unconventional, actin-associated motor protein Myosin1d (Myo1d) as well as the transcription factor Doublesex and mab-3 related 2 (Dmrt2) intervene in LR specification was analyzed in this chapter. In evolutionary terms the study of myo1d was of high interest because in Drospohila, which lacks a ciliary flow mechanism, the homologous gene, myo31df, controls LR axis determination. Manipulations of myo1d in Xenopus demonstrated that in vertebrates Myo1d is involved in the cilia-based symmetry breakage event. By interacting with the PCP signaling pathway, Myo1d ensures leftward-fluid flow by regulating ciliary outgrowth and polarization. In Drosophila and Xenopus Myo1d interacts with PCP signaling and seems to link an ancestral symmetry breaking mechanism of the fly to the newly evolved leftward-fluid flow in vertebrates. Based on studies in zebrafish, which identified Dmrt2 as another factor involved in LR development and somitogenesis, we started the analysis of dmrt2 in Xenopus. Somitogenesis and laterality determination which on first sight are functionally distinct processes were analyzed in the context of dmrt2 function. In Xenopus, flow-sensing cells are affiliated to the somitic cell lineage and therefor paraxial mesoderm specification is crucial for setting up a functional LRO. Dmrt2 specifies the paraxial mesoderm and especially the sLRO by inducing the myogenic transcription factor myf5 in early gastrulae. This demonstrated for the first time experimentally how somitogenesis and laterality determination are intertwined and describes the genesis of the Xenopus sLRO cells in more detail.Während der frühen Embryogenese generieren embryonale Musterbildungsprozesse entlang der Kopf-Rumpf- (anteroposterior, AP), Rücken-Bauch- (dorsoventral, DV) und links-rechts (LR) Körperachse den grundlegenden Bauplan der Bilateria. Hierbei ist vor allem die Ausbildung der LR-Achse auffallend: sie besticht durch eine äußerlich sichtbare Symmetrie entlang der AP-Achse, wohingegen die asymmetrische Formgebung und Position der inneren Organe in der sekundären Leibeshöhle äußerlich nicht zu erkennen ist. Die Ausbildung der drei Körperachsen wird durch die Aktivität zahlreicher Gene während der Gastrulation und Neurulation reguliert. Ziel dieser Arbeit war es, die Rolle der hoch konservierten Gene goosecoid (gsc), myosin1d (myo1d) und doublesex-and mab3 related transcription factor 2 (dmrt2) während der Ausbildung der Körperachsen in Xenopus laevis näher zu untersuchen. Das erste Kapitel dieser Arbeit befasst sich mit der frühen Funktion des Homöobox-Transkriptionsfaktors Goosecoid während der Ausbildung der AP- und DV-Achse. Gsc wirkt als autoregulatorischer transkriptioneller Repressor, wird im Spemann-Organisator, dem Signalzentrum der primären Achseninduktion exprimiert und steuert die embryonale Musterbildung. Es reprimiert ventrale Signalwege im dorsalen Gewebe, separiert das Kopf- vom Chordamesoderm und reguliert Zellbewegungen im Zuge der Gastrulation und Neurulation. Die frühe Funktion von gsc im Spemann-Organisator war bislang enigmatisch, da der Funktionsverlust von gsc die frühe embryonale Entwicklung nicht beeinträchtigte. Durch gezielte Überexpression von gsc in der dorsalen Mittellinie von Xenopus Embryonen wurde hier die frühe Funktion von gsc näher charakterisiert. Gsc agierte sowohl zell- als auch nicht-zell-autonom als Repressor planarer Zellpolarität (planar cell polarity, PCP). In der frühen Gastrula separierte Gsc durch die Repression des mesodermalen T-box Gen Transkriptionsfaktors T (Tbxt) das Kopf- vom Chordamesoderm. Dies ermöglichte das migrieren des Kopfmesoderms und beschränkte die durch Tbxt-induzierte PCP-vermittelte mediolaterale Interkalation auf das elongierende Notochord des Embryos. Diese Funktion von Gsc scheint sich im Zuge der Evolution durch die Etablierung zweier neuer, für Vertebraten spezifische Domänen etabliert zu haben. Das zweite Kapitel befasst sich mit der Determinierung der LR-Körperachse in Xenopus, die als letzte der drei Körperachsen festgelegt wird. Diese wird durch einen Cilien-basierten nach links-gerichteten Flüssigkeitsstrom innerhalb des sog. links-rechts Organisators (LRO) in der Neurula initiiert. Die lateralen, linken sensorischen Zellen des LROs (sLRO) perzipieren den Flüssigkeitsstrom und translatieren dieses Signal in die Induktion der hoch konservierten Nodal Kaskade auf der linken Seite. Welche Funktion das unkonventionelle, Aktin-assoziierte Motorprotein Myo1d und der Transkriptionsfaktor Dmrt2 bei diesem Prozess einnimmt, wurde im Rahmen dieser Arbeit untersucht. Die Analyse von myo1d war hierbei evolutionär von großer Bedeutung, da das homologe Gene myo31df in Drosophila die Entstehung der LR-Achse, unabhängig eines links-gerichteten Flüssigkeitsstrom und einer asymmetrischen Gen-Kaskade reguliert. Die Manipulation von myo1d in Xenopus demonstrierte, dass die Funktion von Myo1d konserviert ist und auch in Vertebraten für den Symmetriebruch benötigt wird. Durch Interaktion mit dem PCP Signalweg trägt Myo1d über die Polarisierung und Ausbildung der Cilien zum links-gerichteten Flüssigkeitsstrom und somit zur Lateralitätsdeterminierung in Xenopus bei. Durch den Einfluss von Myo1d auf die PCP in Drosophila und Xenopus stellt Myo1d eine direkte Verbindung zwischen dem ancestralen Mechanismus und des in Vertebraten neu-evolvierten Flüssigkeitsstrom zum Bruch der bilateralen Symmetrie dar. Studien aus dem Zebrabärbling identifizierten Dmrt2 als einen weiteren Faktor, der sowohl für die Somitogenese als auch für die Ausbildung der LR-Körperachse benötigt wird. Ein Zusammenhang zwischen diesen Prozessen ist ein lang bekanntes Phänomen, dessen Ursache bisher nicht geklärt wurde. Aufgrund der Integration der sLRO Zellen in das paraxiale presomitische Mesoderm, dem Vorläufergewebe der Somiten, stellte sich die Frage, ob dies eine Verbindung zwischen diesen zwei Prozessen erklären könnte. Die Untersuchung von Xenopus Embryonen nach Manipulation von dmrt2 zeigte, dass die Spezifizierung des paraxialen Mesoderms in der frühen Gastrula für die Ausbildung der sLRO Zellen ausschlaggebend ist. Über die Induktion des myogenen Transkriptionsfaktors myf5 reguliert Dmrt2 die Spezifizierung des paraxialen Mesoderms und ins Besondere der sLRO Zellen in Xenopus. Dies demonstrierte zum ersten Mal experimentell eine direkte Verbindung zwischen der frühen Somitogenese und der Lateralitätsdeterminierung und liefert eine erste Erklärung wie diese Prozesse zusammenhängen